1. Field of the Invention
The present invention relates to the field of cell isolation, including the isolation of cells from peripheral circulation, labeling cells of interest magnetically, and using an automatable apparatus to immobilize and isolate viable labeled cells for further testing and culture.
2. Related Art
Background
Utilizing current technologies, it is possible to count the number of circulating tumor cells present in blood from breast cancer patients and predict disease outcome [1,2]. However, due to the lack of additional information about the population of circulating tumor cells, current methods do not offer insights into directing treatment or developing novel therapies. Current targeted treatments based on breast cancer subtypes, e.g., Her2/neu or estrogen receptor (ER) status, only focus on the subtype of the primary tumor [3,4,5]. Recent studies have shown that a portion of breast cancer metastases have different Her2 and ER status compared to the original primary tumors [6,7,8,9]. By analyzing the circulating tumor cells (CTCs) for their genetic characteristics, one can target treatment not only to the primary tumor, but also to cells that may contribute to and serve as surrogate markers of metastases, thus improving survival in women with metastatic disease.
CTCs can be collected through a relatively non-invasive blood draw. However, isolating, purifying, and characterizing these cells has proven challenging. Several current technologies allow for isolation and counting of circulating tumor cells from patient blood samples. Additionally, the expression of two or three biomarkers can also be assessed. However, while the CTCs are enriched during these protocols, they are often heavily contaminated with blood cells, may not be viable, or the RNA may be severely compromised, making it difficult to reliably measure gene expression or simultaneously measure the expression of large numbers of genes. Described below is a robotic device which allows one to obtain completely purified, living CTCs. In combination with multiplex qRT-PCR, genes from single CTCs isolated from the blood of patients with metastatic breast cancer were analyzed for expression levels. Thus, current techniques partially purify CTCs from blood, but there is still residual contamination with other blood cells. Some techniques also fix and permeabilize the CTCs, making them unsuitable for downstream microarray analysis or in vitro and in vivo biological studies.
CTC biology is still poorly defined because most studies assess only CTC burden. The characterization of CTCs is a nascent field; isolation of CTCs specifically for multigene molecular analyses, in contradistinction to counting, is challenging for multiple biological and technical reasons. The cells are fragile, likely a result of mechanical stresses on CTCs in the blood stream and chemical effects of cytotoxic chemotherapy. Moreover, CTCs are extremely rare. For example, 81% of metastatic breast cancer patients will have less than ten CTCs in a 7.5 cc tube of blood containing about 1010 blood cells. Technical factors that impede CTC gene expression analysis in currently available platforms include cell fixation and permeabilization, immobilization, and, most importantly, blood cell contamination. CTC fixation and permeabilization performed prior to fluorescent labeling of CTCs can structurally modify RNA and impact cell viability. CTC immobilization on substrates such as glass slides, filters, or microposts limit single cell manipulation. Finally, even after enrichment, CTCs may be contaminated by thousands of leukocytes (white blood cells, WBCs) that confound expression analysis, requiring bioinformatic techniques to subtract non-CTC gene expression. Thus, direct simultaneous analysis of many gene targets in single human CTCs has yet to be performed.
Certain commercial technologies use immunomagnetic enrichment. Commercial products include the CellTracks® AutoPrep® System and CellSearch™ Circulating Tumor Cell Kit (Immunicon Corporation, Huntingdon Valley, Pa.), MACS® separation technology (Miltenyi Corporation, Bergisch Gladbach, Germany), and the RoboSep® automated cell separator (StemCell Technologies, Vancouver, Canada). With these techniques, the epithelial cells in the blood are labeled with magnetic particles attached to an antibody targeted to an epithelial cell surface marker, usually EpCAM. The blood is processed and external magnets hold the epithelial cells at the side of the tube, while the other blood cells are diluted and pipetted out or eluted through a column. The remaining epithelial cells are then available for immunocytochemical analysis, again amidst 1000-10,000 WBCs. Because of heavy mononuclear cell contamination (whose nucleic acids or proteins would overwhelm any subsequent molecular analyses of the CTCs), most analyses stain and count cells, or limit characterization to one to two immunostains.
Specific Patents and Publications
U.S. Pat. No. 3,970,518 to Giaever, issued Jul. 20, 1976, entitled “Magnetic separation of biological particles,” discloses a method and apparatus in which the particular cell population that is to be separated from a mixed population is contacted with small magnetic particles or spheres which are first provided with a monomolecular coating of antibody to this select population. As the metallic particles enter the field created by a coil at the bottom of the vessel, they are captured and immobilized while liquid is unaffected and leaves vessel.
U.S. Pat. No. 7,125,964 to Luxembourg, et al., issued Oct. 24, 2006, entitled “Purification of antigen-specific T cells,” discloses a method to capture, purify and expand antigen-specific T lymphocytes, using magnetic beads coated with recombinant MHC class I molecules. The inventors used attachment of biotinylated MHC Class I molecules on Aaidin-coated magnetic beads.
U.S. Pat. No. 5,200,084 to Liberti, et al. Apr. 6, 1993, entitled “Apparatus and methods for magnetic separation,” discloses a magnetic separation apparatus and methods for separating colloidal magnetic particles from a non-magnetic test medium in which the magnetic particles are suspended. The separator comprises a container holding the non-magnetic test medium, one or more magnetic wires disposed substantially within the test medium in the container and an external magnet (illustrated at 31 of FIG. 1 of the patent) for producing a magnetic field gradient within the test medium. According to the method of the invention, the container holding the test medium is positioned in the separator, producing a magnetic field gradient operative to cause the magnetic particles to be attracted to the areas surrounding the magnetized wires and to adhere to the wires.
U.S. Pat. No. 5,837,144 to Bienhaus, et al., issued Nov. 17, 1998, entitled “Method of magnetically separating liquid components,” discloses Method of separating a component of a liquid from other components by immobilizing the component to suspended magnetic particles in a vessel, immersing a magnetic device into the vessel while the device is separated from the liquid by means of a protective sleeve made of a non-magnetic material. The protective sleeve is selected such that its outer surface is always spaced apart from the inner surface of the vessel by approximately the same distance.
U.S. Pat. No. 6,468,810 to Korpela, issued Oct. 22, 2002, entitled “Magnetic particle transfer device and method,” discloses a pipette like device for transfer suitable for capturing and releasing microparticles binding an immobilized substance, which includes a magnet as well as either an extendable membrane, shapable membrane or magnet's coating such that the membrane or coating pressing tightly against the magnet's surface separates the magnet from the microparticles but does not substantially weaken the magnetic field directed at the microparticles (See FIGS. 1D and 1E of the patent).
US 20070251885 by Korpela et al., published Nov. 1, 2007, entitled “Method and a Device for Treating Microparticles,” disclose a method for handling microparticles in such a manner, that at least two treatment steps are performed for microparticles in the same vessel without moving the particles to another vessel. This can be brought about by moving the magnet inside the ferromagnetic tube in such a manner, that it can be completely inside the tube, whereupon the efficiency of the magnet is insignificant or nonexistent, or it can be partially or completely outside the tube, whereupon the efficiency and the collecting area of the magnet are in relation to the protruding part of the magnet.